Pseudomonas aeruginosa is an opportunist pathogen capable of infecting a number of tissues in the human body. Its ability to cause a wide-range of infections and its resistance to antimicrobials has made P. aeruginosa one of the most threatening pathogens facing the world today. Notably, P. aeruginosa produces numerous virulence factors that lead to extreme tissue injury, and it is the long-term goal of the proposed project to combat the production of these virulence factors by antagonizing genome-wide transcription from genes regulated by the sigma factor RpoN and transcription factors known as enhancer-binding proteins (EBPs). RpoN and EBPs interact with one another to activate transcription of target genes in response to various signals. In some instances, histidine kinases sense the signals and relay that information to EBPs via phosphorylation. The overall objective of the proposed project is to characterize the EBPs and their partner histidine kinases in P. aeruginosa. Knowledge of this regulation, including the signals, target genes and regulatory mechanisms is essential to achieve the long-term goal of the proposed project. The central hypothesis is that EBPs and their partner HKs regulate an assortment of functions crucial to P. aeruginosa pathogenesis, including the utilization of various host-derived nutrients, protein secretion, production of virulence factors and alginate biosynthesis. The rationale for the proposed project is that complete knowledge of EBP regulation, i.e., the signals and target genes of EBP regulation, will provide the information needed to develop a strategy to counter P. aeruginosa pathogenesis via specifically attacking RpoN-EBP regulation. Two specific aims are proposed to test the central hypothesis. In Specific Aim 1, EBPs that have not yet been biochemically characterized (AauR, MifR, DdaR and EatR) or have unknown functions (PA1663 and PA1945) will be investigated. The DNA-binding specificities will be measured for each EBP, and in the case of PA1663 and PA1945, transcriptome studies will be performed to identify their potential target genes and biological functions. In Specific Aim 2, the substrate specificities and kinetics will be measured for EBP-related histidine kinases with poorly defined mechanisms, including AauS, CbrA, DctB, KinB and MIfS. Signals or cues that stimulate the activities of these histidine kinases (and thus EBP regulation) will be identified. The approach is innovative because it will establish a complete, working model for RpoN-EBP regulation in P. aeruginosa, and importantly, define how this regulation is connected to bacterial pathogenesis on a molecular level. The proposed project is significant, because knowledge of this regulation will enable the development of antibacterial agents or compounds that can be used to combat the pathogenesis of P. aeruginosa.